LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA

ABSTRACT Objective: This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Materials and methods: Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. Results: XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory response in HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. Conclusions: LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.


INTRODUCTION
D iabetic retinopathy (DR) is a microvascular complication of diabetes, leading to specific changes in the fundus of the eye (1).The global prevalence of DR is reported to be 22.27% among patients with diabetes in 2019 (2).When DR progresses at the proliferative (advanced) stage, the retina develops new blood vessels, resulting in vitreous/pre-retinal hemorrhage and tractional retinal detachment (3).With regard to the pathology, hyperglycemia-induced metabolic abnormalities stimulate oxidative stress, which causes inflammation and regulates neovascularization in the retina (4).Vascular endothelial growth factor (VEGF) is known as an established molecule in the pathogenesis of DR and intravitreal administration of agents antagonizing VEGF shows favorable outcomes in the management of DR (5).VEGF exerts a vital role in mediating the activities of several kinases and ultimately drives cell proliferation, migration, and vascular permeability during vascularization (6).A comprehensive and profound understanding of the complicated mechanisms underlying the regulation of VEGF may provide a basis for optimizing the therapeutic effect of anti-VEGF therapy on DR.
The human genome expresses a mass of long noncoding RNAs (lncRNAs) that confer diverse roles in cell biology and human disease at epigenetic, transcriptional, and translational levels (7).Recent investigation has come to light indicating the role of several lncRNAs in regulating endothelial function in DR, such as MEG3, VEAL2, and MALAT1 (8)(9)(10).LncRNA X inactive-specific transcript (XIST), which is transcribed from the XIST gene responsible for X chromosome inactivation, is shown to regulate retinal glial and epithelial cells under hyperglycemia (11,12).Moreover, lncRNA XIST maintains VEGF signaling in human brain microvascular endothelial cells and is required for hypoxia-induced angiogenesis (13).
In light of the above evidence, we hypothesized that XIST might maintain VEGFA expression by targeting miR-101-3p, thereby driving retinal angiogenesis in DR.This study was conducted to dissect the function of the XIST/miR-101-3p/VEGFA axis in DR for the purpose of uncovering new molecular mechanisms underlying the pathogenesis of DR, hoping to provide a reliable theoretical basis for exploring potential targets for the treatment of DR.

Clinical samples
This study enrolled 20 patients with DR (13 males and 7 females, aged 59.21 ± 9.57 years) who visited the Department of Ophthalmology in Ningbo Medical Center Lihuili Hospital from August 2021 to January 2022.Additionally, another 20 patients with diabetes but without retinopathy (11 males and 9 females, aged 56.11 ± 9.79 years) were included.The basic characteristics of all patients are listed in Table 1.The patients with DR were included according to the WHO diagnostic criteria for diabetes (20) and diagnostic criteria for DR (21).None of the participants had cardiovascular diseases, peripheral vascular diseases, liver or kidney dysfunction, or malignancy.Venous blood samples were collected after 12-h fasting.This study was approved by the Ethics Committee of Ningbo Medical Center Lihuili Hospital and totally complied with the Declaration of Helsinki.All patients had signed the written informed consent.
The control group was cultured with 5.5 mM glucose (Sigma-Aldrich, St. Louis, MO, USA), while the HG group was treated with 25 mM glucose for 48 h.

qRT-PCR
The isolation of total RNA was conducted with TRIzol and the concentration was subsequently determined using a spectrometer (DU-640; Beckman, San Jose, CA, USA).The RT kits (RR047A; Takara, Japan) and miRNA first strand cDNA synthesis (tailing reaction) kits (B532451-0020; Sangon, Shanghai, China) were used to synthesize cDNA for detection of mRNA and miRNA, respectively.Gene expression was examined using SYBR Green Mix (HY-K0501A; MedChemExpress, Monmouth Junction, NJ, USA) and LightCycler 480, with a reaction volume of 20 μL and the following parameters: 95 °C (10 s); 30 cycles

Transwell detection of cell migration
Cells in the exponential growth stage were trypsinized and then diluted to a concentration of 1 × 10 5 cells/mL with the 1% FBS-complete medium.Cell suspension (200 μL) was transferred to each upper chamber of a 24-well plate equipped with transwell inserts, and 10% FBS-medium (500 μL) was added to the lower chamber.After 48-h culture, non-invasive cells were removed, and the insert membrane was washed twice with PBS.Invasive cells were treated with 5% paraformaldehyde and 0.5% crystal violet for 15 min each, and the number of cells passing across the membrane was counted under an Olympus microscope (CKX53).

Tube formation assay
The assay was carried out as previously described (23).Matrigel (Corning, Tewksbury, MA, USA) was pre-dissolved at 4°C, and 96-well plates (Millipore, Billerica, MA, USA) and pipette tips were pre-chilled.Each well was plated with 100 μL of Matrigel, and the plate was then placed in an incubator for 30 min to solidify the Matrigel.HRMECs were seeded in the 96-well plates pre-coated with Matrigel (2 × 10 4 cells/ well, 3 replicate wells/sample).The cells were routinely cultured for 18 h and photographed under a CKX53 microscope.A tube was considered a tubular structure that extended from one branching point to another or to a loose end.Capillaries in 3 visual fields/group were counted using Image-Pro Plus 6.0 software.

ELISA
The levels of TNF-α, IL-6, and IL-1β were detected in conformity with the provided manuals of ELISA kits (R&D Systems, UK).Samples were pre-incubated in an ELISA plate (96 wells) overnight at an ambient temperature.Thereafter, 100 μL of 5% BSA was added to each well to block nonspecific binding for 60 min.The samples were incubated with the primary antibody (diluted in 5% BSA-PBS, 100 μL/well) for 3 h and next with the HRP-labeled secondary antibody (diluted in Table 2. PCR primers and their sequences

Dual-luciferase reporter assay
The binding sites between miR-101-3p with lncRNA XIST and VEGFA were predicted by starBase (http:// starbase.sysu.edu.cn/).A wild-type sequence (wt-XIST or wt-VEGFA) or mutant-type sequence (mut-XIST or mut-VEGFA) of the predicted binding sites was cloned to the pGL3-Basic and co-delivered with 30 nM miR-101-3p or NC mimic into the 293T cells.Following 48-h transfection, luciferase activity was detected using a detection kit (Promega, Madison, WI, USA) and analyzed using a Promega system.

RNA pull-down
The Pierce TM magnetic RNA-protein pull-down kit (Millipore) was utilized in our assay.Biotinylated NC or miR-101-3p probes (Geneseed, Guangzhou, China) were incubated with cell lysates for 120 min at 25 °C.Immune complexes were pulled down by streptavidintagged immunomagnetic beads and treated in proteinase K buffer for 60 min at 25 °C respectively, followed by qRT-PCR analysis of the eluted RNA.

RNA immunoprecipitation (RIP)
Magnetic beads were suspended in the RIP wash buffer (100 μL) and kept with anti-Ago2 (ab186733) or anti-IgG (ab172730) antibody (both 1:100 and 5 μg; Abcam) at an ambient temperature for 0.5 h.The bead tube was placed on a magnetic base, and then the supernatant was removed.The complexes were washed twice with RIP wash buffer (500 μL).The prepared bead tube was placed on the magnetic base, the supernatant was removed, and RIP immunoprecipitation buffer (900 μL) was added.The complexes were kept with cell lysate 100 (μL) overnight.After a brief spin, magnetic precipitation, and six washes, the complexes were incubated with proteinase K buffer 150 (μL) for 0.5 h at 55 °C.The tube was then put on the magnetic base, and the supernatant was extracted for qRT-PCR RNA detection.

Animal grouping and treatment
The 30 SD rats were grouped as control, DR, sh-XIST+oe-NC, sh-XIST+oe-VEGFA, and sh-NC+oe-NC.In the latter three groups, 1 μL of lentiviruses (10 9 PFU/mL (24,25)) as indicated in their group names were injected into the vitreous cavity of rats on the 7th day after successful induction of diabetes.Subsequent experiments were performed 4 weeks after lentiviral injection.

Statistical analyses
Statistics were analyzed with GraphPad Prism 8, and all data were depicted as mean ± SD.All cell experiments were conducted in triplicate.Two groups were compared using t-test, and one-way analysis of variance was implemented for comparisons among three or more groups, followed by Tukey's multiple comparisons test.P < 0.05 represented a statistically significant difference.

Low XIST expression inhibits angiogenesis and inflammatory response in HG-treated HRMECs
First, qRT-PCR was carried out to detect XIST expression in patients with diabetes or DR, which revealed remarkable upregulation of XIST expression in patients with DR (Figure 1A, * P < 0.05).Consistently, XIST expression was increased in HG-treated HRMECs, the cellular models of DR (Figure 1B, * P < 0.05).Next, to examine the function of XIST in DR, the silencing vector sh-XIST was utilized to induce low expression of XIST in HG-treated HRMECs (Figure 1B, # P < 0.05).MTT and transwell assays were conducted to assess cell viability and migration, respectively, which showed that HG treatment stimulated the proliferation and migration of HRMECs (Figure 1C, D, * P < 0.05).
The levels of these cytokines declined upon sh-XIST interference (Figure 1F, # P < 0.05).Consequently, downregulation of XIST inhibits angiogenesis and inflammatory response in HG-treated HRMECs.

VEGFA is targeted by miR-101-3p in DR
The TargetScan database predicted the binding sites in VFGFA for miR-101-3p (Figure 4A).Therefore, we hypothesized that XIST may regulate VFGFA via miR-101-3p to affect angiogenesis and inflammatory response in HRMECs.We found that the mRNA and protein levels of VFGFA were elevated in patients with DR as well as HG-treated HRMECs (Figure 4B-E

DISCUSSION
The number of people with DR keeps rising worldwide with the increasing prevalence of diabetes and prolonged lifetime of patients with diabetes (26).
VEGFA is regarded as a predominant pathogenic factor in regulating neovascularization in DR, and intravitreal administration of anti-VEGF molecules has radically improved the management of DR (27).Increasing the duration of anti-VEGF therapy and developing more optimized molecules are hot issues in this field.This study elucidated upregulated XIST and VEGFA and downregulated miR-101-3p in patients with DR.XIST indirectly upregulated VEGFA by targeting miR-101-3p, thereby stimulating angiogenesis in HG-treated HRMECs.
Many studies have been conducted to address the mechanisms underlying the regulation of VEGF in DR.For example, berberine, an isoquinoline alkaloid, is known to inactivate the Akt/mTOR signaling in retinal endothelial cells to inhibit insulin-induced activity of hypoxia-inducible factor-1α and VEGF (28).miR-21 activates the phosphatidylinositiol 3-kinase/Akt/ VEGF signaling pathway by targeting phosphatase and tensin homolog, thereby stimulating vascular  endothelial cell viability and angiogenesis in the retina of rats with diabetes (29).Circular RNA COL1A2 contributes to HG-induced proliferation, migration, and vascular permeability in HRMECs and retinal angiogenesis in mice with diabetes by competing with VEGF mRNA for binding to miR-29b (30).There are many other miRNAs, such as miR-15b, miR-23a, miR-205-5p, and miR-150-5p, which directly interact with VEGF in retinal endothelial cells and are involved in angiogenesis in DR (31)(32)(33)(34).This study identified miR-101-3p as a direct regulator of VEGFA in HRMECs.
As previously reported, miR-101-3p suppresses the release of VEGFA from cancer-associated fibroblasts by targeting VEGFA mRNA and subsequently reduces migration and invasion of non-small cell lung cancer cells (19).Although the function of miR-101-3p in HRMECs has not been reported to date, there are several studies indicating the implications of miR-101-3p for vascular endothelial function.For example, compelling evidence suggests that miR-101-3p overexpression promotes ROS production and induces cytoskeletal destruction in human umbilical vein endothelial cells by targeting TET2 (35).Zika virus could upregulate hsa-miR-101-3p in human brain microvascular endothelial cells to suppress VE-cadherin and claudin-5, two endothelial barrier integrityresponsible factors (36).Moreover, miR-101-3p expression is downregulated to promote endothelialmesenchymal transition and renal fibrosis in mice with diabetes (37), suggesting the involvement of miR-101-3p in the regulation of endothelial function in diabetes.Many lncRNA-miRNA interplays are involved in the development of DR (38).Consistent with previous findings about the interaction of lncRNA XIST with miR-101-3p (16,17,39), this study established the XIST/miR-101-3p axis in HRMECs.
Current knowledge further indicates that HGinduced downregulation of XIST increases apoptosis and reduces migration in human retinal pigment epithelial cells by upregulating hsa-miR-21-5p expression (11).Moreover, XIST is downregulated to facilitate HG-induced activation of retinal Müller cells and production of pro-inflammatory cytokines (12).However, evidence favoring the function of XIST in HRMECs in DR is sparse.Wang and cols.have found that XIST silencing impedes angiogenesis and exacerbates cerebral vascular injury in ischemic stroke by regulating proangiogenic integrin α5 and anti-inflammatory Krüppel-like transcription factor 4 via miR-92a (40).In addition, XIST upregulation protects brain microvascular endothelial cells from pyroptosis following ischemic injury (41).In this study, low XIST expression was uncovered to repress proliferation, migration, angiogenesis, and secretion of pro-inflammatory cytokines in HG-treated HRMECs.
Accumulating evidence has suggested that VEGFA can be regulated by the lncRNA/miRNA network in DR.For instance, linc00174 could deteriorate diabetic retinal microangiopathy by modulating the miR-150-5p/VEGFA pathway (33).Malat1 knockdown suppresses the release of VEGFA by targeting miR-205-5p to curb HRMEC growth and tube formation under HG conditions (32).Mechanistically, TUG1 acts as a competing endogenous RNA for miR-145 to promote VEGFA expression in HRMECs, and repression of miR-145 annuls the beneficial roles of TUG1 silencing in HG-stimulated HRMECs (42).However, there is no report regarding the action of the XIST/miR-101-3p/VEGFA axis in HRMECs.Therefore, we then conducted a series of experiments with anticipation to fill this knowledge gap.Similar to the previously reported lncRNA/miRNA/VEGFA axis, our results elucidated that downregulation of miR-101-3p or overexpression of VEGFA neutralized the anti-angiogenic and anti-inflammatory effects of XIST knockdown on HG-treated HRMECs.An existing research has pointed out the constant high abundance of miR-101 in developing rat retinas (43), suggesting the essential role of miR-101 in the development of retinas.Intrinsically, the upregulation of VEGFA is shown to disrupt the retinal barrier, contributing to chronic damage to the neurovascular structure of the retina, ultimately causing vision loss (44).VEGFA overexpression confers pro-migratory, pro-proliferative, and pro-angiogenic actions in HG-treated HRMECs (45,46).Subsequently, animal experiments were performed for in vivo verification of this mechanism.As wellknown, disruption of the blood-retinal barrier (BRB) is the pathophysiological basis for increased vascular permeability and macular edema in DR and is an important cause of vision loss in patients with DR (47).BRB integrity is determined by junctional complexes composed of tight junctions and adherent junctions, and cell-to-cell connection is composed of tight junctions, adherent junctions, and desmosomes, among which tight junctions are responsible for the barrier function between cells (48).The tight junctions are the basis of the BRB and are essential for maintaining the structural and functional integrity of the BRB as well as the stability of the intraretinal environment.The BRB tight junctions are mainly composed of transmembrane proteins, cytoplasmic adhesion proteins, and cytoskeletal proteins.The transmembrane proteins are mainly composed of claudins, occludins, and ZO-1 (48).Therefore, the expression levels of permeabilityrelated proteins (ZO-1, claudin-5, and occludin) can be determined to evaluate retinal lesions.Consistent with the findings obtained from cell experiments, downregulation of XIST reduced the histopathological changes and levels of TNF-α, IL-1β, IL-6, and meanwhile upregulated ZO-1, claudin-5, and occludin levels in the retina of rats with DR, but the above effects were counteracted by VEGFA overexpression.Collectively, the aforementioned findings and evidence underscore that XIST drives angiogenesis and inflammatory response in DR by upregulating VEGFA expression via miR-101-3p.

Figure 1 .
Figure 1.Low expression of XIST inhibits angiogenesis and inflammatory response in HG-treated HRMECs.(A) qRT-PCR was used to detect XIST expression in patients with diabetes or DR (n = 20).HRMECs were treated with HG and transfected with sh-NC or sh-XIST.Next, (B) qRT-PCR was performed to detect XIST expression, (C) MTT assay to detect cell proliferation, (D) transwell assay to detect cell migration, (E) tube formation assay to detect angiogenesis, and (F) ELISA to detect levels of inflammatory factors in culture supernatant.The data were expressed as mean ± standard deviation.Each cell experiment was repeated thrice.* P < 0.05, compared with the NDR or control group; # P < 0.05, compared with the sh-NC group.

Figure 2 .
Figure 2. XIST targets miR-101-3p in DR. (A) The binding sites and corresponding mutations of miR-101-3p and XIST.qRT-PCR was used to detect miR-101-3p expression in patients with DR (B) and HG-treated HRMECs (C).(D) Pearson's correlation analysis of the expression of XIST and miR-101-3p in patients with DR.Dual-luciferase reporter (E) and RIP (F) assays were performed to verify the binding of miR-101-3p and XIST.(G) qRT-PCR was used to detect the expression of miR-101-3p in HRMECs transfected with oe-NC, oe-XIST, sh-NC, or sh-XIST.The data were expressed as mean ± standard deviation.The clinical sample size was 20.Each cell experiment was repeated thrice.* P < 0.05, compared with the NDR or control group; # P < 0.05, compared with the mi-NC, oe-NC, or IgG group; & P < 0.05, compared with the sh-NC group.

Figure 3 .
Figure 3. Downregulation of miR-101-3p reverses anti-angiogenic and anti-inflammatory effects of XIST knockdown on HG-treated HRMECs.HRMECs were treated with HG and transfected with sh-NC + in-NC, sh-XIST + in-NC, or sh-XIST + in-miR-101-3p.Next, (A) qRT-PCR was performed to detect miR-101-3p expression, (B) MTT assay to detect cell proliferation, (C) transwell assay to detect cell migration, (D) tube formation assay to detect angiogenesis, and (E) ELISA to detect levels of inflammatory factors in culture supernatant.The data were expressed as mean ± standard deviation.Each cell experiment was repeated thrice.* P < 0.05, compared with the sh-NC+in-NC group; # P < 0.05, compared with the sh-XIST+in-NC group.

Figure 4 .
Figure 4. miR-101-3p targets VEGFA in DR. (A) The miR-101-3p-binding site in VEGFA and corresponding mutations.qRT-PCR and western blotting were used to detect the mRNA expression and protein level of VEGFA in patients with DR (B, C) and HG-treated HRMECs (D, E). (F) Pearson's correlation analysis of the expression of VEGFA and miR-101-3p in patients with DR.RNA pull-down (G) and dual-luciferase reporter (H) assays were performed to verify the binding of miR-101-3p and VEGFA.qRT-PCR (I) and western blotting (J) were used to detect the mRNA expression and protein level of VEGFA in HRMECs transfected with mi-NC, mi-miR-101-3p, in-NC, or in-miR-101-3p.The data were expressed as mean ± standard deviation.The clinical sample size was 20.Each cell experiment was repeated thrice.* P < 0.05, compared with the NDR, control, or mi-NC group; # P < 0.05, compared with the NC probe or in-NC group.

Figure 5 .
Figure 5. VFGFA overexpression reverses the anti-angiogenic and anti-inflammatory effects of XIST knockdown on HG-treated HRMECs.HRMECs were treated with HG and transfected with sh-NC + oe-NC, sh-XIST + oe-NC, or sh-XIST + oe-VFGFA.Next, (A) qRT-PCR was performed to detect VFGFA mRNA expression, (B) western blotting to detect VFGFA protein expression, (C) MTT assay to detect cell proliferation, (D) transwell assay to detect cell migration, (E) tube formation assay to detect angiogenesis, and (F) ELISA to detect levels of inflammatory factors in culture supernatant.The data were expressed as mean ± standard deviation.Each cell experiment was repeated thrice.* P < 0.05, compared with the sh-NC+oe-NC group; # P < 0.05, compared with the sh-XIST+oe-NC group.

Figure 6 .
Figure 6.XIST promotes retinal angiogenesis and inflammatory response in rats with DR through the miR-101-3p/VEGFA axis.DR model rats were injected with sh-NC + oe-NC, sh-XIST + oe-NC, or sh-XIST + oe-VFGFA.(A, B) qRT-PCR was used to detect the expression levels of XIST, miR-101-3p, and VEGFA mRNA in retinal tissue.(C) Western blotting was used to detect the expression of VEGFA protein in retinal tissue.(D) H&E staining was used to detect pathological changes in the retina.(E) ELISA was performed to detect the levels of TNF-α, IL-1β, and IL-6 in retinal tissue.(F) Western blotting was used to detect the expression levels of ZO-1, claudin-5, and occluding in retinal tissue.The data were represented by mean ± standard deviation.Each group had 6 rats.* P < 0.05, compared with the normal group; # P < 0.05, compared with the sh-NC+oe-NC group; & P < 0.05, compared with the sh-XIST+oe-NC group.